Bradykinin is an endothelium-dependent vasodilator and inflammatory mediator. The aim of the present study was to examine the effects of bradykinin on peripheral vascular tone in patients with cirrhosis and ascites. Forearm blood flow was measured using venous occlusion plethysmography in 8 patients with biopsy-proven alcohol-induced cirrhosis, ascites, and portal hypertension, and 8 age- and sex-matched healthy controls. On 1 occasion, subjects received an intrabrachial infusion of the selective bradykinin antagonist B9340 (1.5–13.5 nmol/min) followed by a control vasoconstrictor norepinephrine (60–540 pmol/min), and on another occasion bradykinin (100–900 pmol/min) followed by the endothelium-independent vasodilator, sodium nitroprusside (SNP, 2–8 μg/min). Bradykinin and SNP caused a dose-dependent vasodilatation (P<0.001 for both) that did not differ between the 2 groups. Although norepinephrine caused a similar dose-dependent vasoconstriction in both groups (P<0.001), B9340 had no effect on forearm blood flow in either group (at 13.5 nmol/min in patients; −5%, 95% CI −13−3). Bradykinin does not provide a major contribution to the maintenance of peripheral vascular tone in patients with cirrhosis and ascites. Our findings also suggest that, in contrast to cardiovascular disease, patients with liver cirrhosis do not have marked endothelial dysfunction.
Departments of *Hepatology
†Cardiology, University of Edinburgh, Royal Infirmary of Edinburgh, Edinburgh, UK
‡Clinical Pharmacology Unit and Research Centre, Department of Medical Sciences, University of Edinburgh, Western General Hospital, Edinburgh
Reprints: James Ferguson, Liver Unit, Royal Infirmary, 51 Little France Crescent, Edinburgh, EH16 4SU, UK (e-mail: firstname.lastname@example.org).
Received for publication September 15, 2005; accepted November 18, 2005
The hyperdynamic circulation is a feature of patients with advanced liver disease and is characterized by low arterial pressure, high cardiac output, splanchnic vasodilatation, and low systemic vascular resistance.1–3 This hyperdynamic circulation is caused by systemic arteriolar vasodilatation, which occurs despite the presence of activated vasopressor systems including the renin–angiotensin and sympathetic nervous systems. It is believed to be responsible for the development of the complications of cirrhosis such as ascites, esophageal varices, and hepatorenal syndrome.4
Bradykinin is produced via the cleavage of high-molecular-weight kininogen by kallikrein.5 Bradykinin causes a powerful endothelium-dependent vasodilatation that is partly mediated through nitric oxide release.6,7 It has a brief duration of action (plasma half-life 15–30 s) because of its rapid degradation by peptidases such as carboxypeptidase N and angiotensin-converting enzyme.7 There are 2 principal kinin receptor subtypes in humans.8,9 The endothelial B2 receptor is constitutively expressed and mediates the profibrinolytic and vasodilatory effects of bradykinin.10,11 The vascular B1 receptor is normally weakly expressed but is upregulated in the presence of inflammation where it also mediates vasodilatation.12
As a potent endothelium-dependent vasodilator, bradykinin may play a role in the hyperdynamic circulation associated with cirrhosis. In a rodent model, exogenous bradykinin causes a B2 receptor-dependent portal hypertension.13 Furthermore, in humans with liver cirrhosis, the kallikrein inhibitor aprotonin caused vasoconstriction and improved renal function.14 However, there are no clinical studies that have directly assessed the contribution of bradykinin to the regulation of vascular tone in patients with cirrhosis. Therefore, the aims of the present study were to examine the effect of exogenous bradykinin and to establish the role of endogenous bradykinin on peripheral vascular tone in patients with cirrhosis and ascites.
MATERIALS AND METHODS
Eight patients with alcohol-induced cirrhosis, ascites, and portal hypertension, and 8 age- and sex-matched healthy subjects were recruited. All of the patients were ambulant, had endoscopically proven varices, and normal serum creatinine (<100 μmol/L). To prevent alcohol-induced depression of vascular responses, patients abstained from alcohol for at least 1 month, confirmed by history and repeated blood ethanol testing.15 To avoid the possibility of altering the endogenous vasopressor systems, all subjects in both groups were maintained on their normal sodium intake (∼150 mmol/day). None of the subjects received vasoactive or nonsteroidal anti-inflammatory drugs the week before, and all abstained from food, tobacco, and caffeine-containing drinks for at least 4 h before each study. All female subjects were postmenopausal both for safety and to avoid the variability in vascular responses that may be associated with cyclical hormonal changes.16 Studies were undertaken in accordance with the Declaration of Helsinki of the World Medical Association, the approval of the local research ethics committee and the written informed consent of each subject.
Intraarterial Drug Administration
Cannulation of the brachial artery of the nondominant arm was performed using a 27 standard wire-gauge steel needle (Cooper's Needle Works, Birmingham, UK) under 1% lidocaine (Xylocaine; Astra Pharmaceuticals, Kings Langley, UK) local anesthesia. Patency was maintained by saline infusion at a constant rate (1 mL/min) via an IVAC P1000 syringe pump (IVAC, Basingstoke, UK).
Pharmaceutical grade B9340 (Clinalfa AG, Läufelfingen, Switzerland), bradykinin (Clinalfa AG, Läufelfingen, Switzerland), sodium nitropusside (SNP; David Bull Laboratories, Victoria, Australia), and norepinephrine (Levophed, Sanofi-Winthrop, Guildford, UK) were dissolved in 0.9% saline (Baxter Healthcare, Thetford, UK) and administered intraarterially. To prevent its oxidation, norepinephrine was dissolved in saline containing 0.1% ascorbic acid (Evans Medical, Langhurst, UK).
All studies were performed in a quiet, temperature-controlled room maintained at 22° to 24°C. Forearm blood flow was measured by venous occlusion plethysmography as described previously.17
Arterial blood pressure and heart rate (HR) were measured in the noninfused arm using a noninvasive oscillometric sphygmomanometer (Takeda UA 751, Takeda Medical, Tokyo, Japan) at 20-min intervals throughout each study. The mean arterial pressure (MAP) was calculated as the diastolic arterial pressure plus one third of the pulse pressure.
Subjects attended on 2 separate occasions at least 1 week apart and rested supine throughout each study. Strain gauges and cuffs were applied and the brachial artery of the nondominant arm was cannulated. In all protocols, saline was infused for 30 min to allow time for equilibration, with forearm blood flow measurements being made every 10 min, and the final measurement taken as the baseline forearm blood flow. On 1 occasion, the endothelium and B2 receptor–dependent vasodilator bradykinin was infused at 100, 300, and 900 pmol/min for 6 min at each dose,10,18 followed by a 30-min saline washout before administering SNP, a control endothelium-independent vasodilator, at 2, 4, and 8 μg/min for 6 min19 at each dose. On the other occasion, B9340, a combined B1 and B2 receptor antagonist, was infused at 1.5, 4.5, and 13.5 nmol/min for 6 min at each dose18,20 followed by a 30-min saline washout before administering norepinephrine, a control vasoconstrictor, at 60, 180, and 540 pmol/min for 6 min at each dose.17
Data Analysis and Statistics
The combination of subsystemic and locally active intrabrachial infusions and bilateral forearm blood flow measurements using venous occlusion plethysmography is a powerful and reproducible method of directly assessing in vivo vascular responses to vasoactive mediators without invoking systemic effects.21 Plethysmographic data were extracted from the Chart (AD Instruments, Castle Hill, Australia) and forearm blood flow was calculated for individual venous occlusion cuff inflations using a template spreadsheet (Excel v5.0; Microsoft, Redmond, WA). The last 5 linear flow recordings in each 3-min period were calculated for each time point, and the noninfused arm used as a contemporaneous control.21 The percentage change in forearm blood flow was calculated as described previously21: Percent change in forearm blood flow=100×[(It/NIt)−(Ib/NIb)]/(Ib/NIb), where It and NIt are the blood flows in the infused and noninfused forearms, respectively, at a given time point (t), and Ib and NIb are the blood flows in the infused and noninfused forearms, respectively, at baseline (b); time 0. Forearm blood flow was expressed in mL×100 mL tissue×min.21
Data were expressed as mean±SE of the mean and examined by analysis of variance (ANOVA) with repeated measures, regression analysis, and 2-tailed paired and unpaired Student t test as appropriate. Statistical significance was taken at the 5% level.
Patients with cirrhosis were well matched to control subjects (Table 1). Throughout each study, there were no significant changes in MAP, HR, or forearm blood flow in the noninfused arm. There was no significant difference in baseline forearm blood flow in the infused arm between patients with cirrhosis and control subjects (3.9±0.7 and 3.9±0.7 mL/100 mL/min, respectively).
Both bradykinin and SNP caused dose-dependent increases in forearm blood flow both in cirrhotic patients and controls (P<0.0001, ANOVA for each group, Fig.'1). The vasodilatation was similar in both groups (P=NS, ANOVA, Fig. 2).
Norepinephrine caused a dose-dependent decrease in forearm blood flow in both groups (P<0.05 ANOVA, Fig.'2). The vasoconstriction was similar in both groups (P=NS, ANOVA, Fig.'2). Bradykinin receptor antagonism during B9340 infusion did not affect blood flow in either group (at 13.5 nmol/min in patients; −5%, 95% CI −13−3, controls; −2%, 95% CI −12−8. P=NS for both, ANOVA).
We have demonstrated that intrabrachial bradykinin infusion causes a similar marked vasodilatation in both patients and matched healthy controls. Despite significant vasoconstriction to norepinephrine, B9340 had no effect on basal forearm blood flow in either group. Our findings suggest that patients with cirrhosis and ascites have normal peripheral endothelium-dependent vasomotor function and that bradykinin does not contribute to basal peripheral vascular tone in patients with cirrhosis.
Bradykinin and Endothelial Function in Cirrhosis
Bradykinin acts on the B2 receptor of the vascular endothelium to release nitric oxide and endothelium-derived hyperpolarizing factor and thereby produce vasorelaxation.6 In the present study, both bradykinin- and SNP-induced vasodilatation were normal in patients with cirrhosis, suggesting that endothelial function is not compromised. This contrasts with a previous study in which methacholine (an endothelium-dependent muscarinic agonist) caused greater forearm vasodilatation in patients with cirrhosis.22 This enhanced activity was not a result of increased vascular smooth muscle cell sensitivity to nitric oxide because endothelium-independent vasodilatation by SNP was unaffected. However, methacholine-induced vasodilatation is not attenuated by nitric oxide synthase inhibition and may not be an ideal investigational agent of nitric oxide–mediated endothelial function.23 The explanation for this enhanced response is also unclear because in classic models of endothelium dysfunction, such as hypercholesterolemia, acetylcholine-induced vasodilatation is reduced not enhanced.24,25 However, Ryan et al previously reported that acetylcholine-induced vasodilatation is impaired in patients with preascitic alcoholic cirrhosis (Childs-Pugh Grade A).26 Taken together, our data suggest that patients with cirrhosis do not have a major impairment in endothelium-dependent vasomotor function in the presence of ascites.
Contribution of Bradykinin to Vascular Tone
B9340 is a selective, reversible, and competitive bradykinin receptor antagonist with a rapid onset and offset of action. At the doses employed in the present study (13.5 nmol/min), we have previously demonstrated that B9340 causes an 18-fold inhibition of bradykinin-induced vasodilatation without affecting the endothelium- and neurokinin type 1 receptor-dependent vasodilatation induced by substance P.27 However, in contrast to patients with heart failure treated with angiotensin-converting enzyme inhibition,27,28 we have found no effect of bradykinin antagonism on basal forearm blood flow in patients with cirrhosis. This suggests that bradykinin does not contribute to basal maintenance of peripheral vascular tone in these patients.
Although bradykinin does not appear to be involved in the maintenance of peripheral vascular tone, this does not preclude a potential role in the vasodilated splanchnic circulation associated with liver cirrhosis. Activation of the kallikrein–kinin system occurs in liver cirrhosis.29 Furthermore, in a rodent model, the portal hypertensive response to bradykinin was shown to be mediated by B2 receptors.13 The authors therefore believe that studies employing systemic bradykinin antagonism are required to determine the role of bradykinin in patients with cirrhosis and portal hypertension.
Previous studies have demonstrated significant differences in vasodilatation in similar population sizes (n=6–12). We can therefore exclude a major difference in bradykinin-induced vasodilatation but accept that our study may have missed a modest effect.
Bradykinin-induced vasodilatation is not only mediated by nitric oxide but also endothelium-derived hyperpolarizing factor, which causes vasodilatation by opening potassium channels in vascular smooth muscle.6,30 Thus, there is a possibility that the normal response to bradykinin seen in the present study may be the result of compensatory changes in vascular mediators such as endothelium derived hyperpolarizing factor. Other agents, such as acetylcholine24 and serotonin,25 induce endothelium-dependent vasodilatation through a mechanism that is either predominantly or completely dependent on nitric oxide release. If there is a specific nitric oxide-mediated vascular dysfunction in patients with cirrhosis, then these agents may be more readily able than bradykinin to demonstrate this impairment. Further research using nitric oxide synthase inhibitors and acetylcholine, serotonin, and cyclooxygenase inhibitors in differing severities of cirrhosis is therefore warranted.
Bradykinin does not contribute to the maintenance of peripheral vascular tone in patients with cirrhosis and ascites. This does not preclude a possible role for bradykinin in the splanchnic circulation but suggests that patients with cirrhosis do not have marked kinin upregulation or endothelial dysfunction in the peripheral circulation.
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Keywords:© 2006 Lippincott Williams & Wilkins, Inc.
cirrhosis; bradykinin; forearm